BD Technical Data Sheet BD Ratiometric Calcium Assay Kit Product Information Catalog Number: 644243 Components: Ratiometric Calcium Indicator, 1 vial, lyophilized 10X Signal Enhancer, 10 ml Calcium Assay Buffer, 90 ml Description The BD TM Ratiometric Calcium Assay Kit allows homogeneous measurement of intracellular calcium changes caused by activation of G-protein coupled receptors or calcium channels. This assay is easy to perform, involving a single dye addition step with no subsequent washing. This makes the Ratiometric Calcium Assay Kit desirable in a high thorough-put environment. Storage Ratiometric Calcium Indicator should be protected from light and stored at -20 C. 10X Signal Enhancer and Calcium Assay Buffer should be stored at room temperature. Materials not included* 100% DMSO Sigma D4540 Probenecid Sigma P8761 * Please see detailed Assay protocol on page 6. 644243 4/24/09 Page 1 of6
CALCIUM ASSAY FLOW CHART 644243 4/24/09 Page 2 of6
DATA EXAMPLES BD TM Ratiometric Calcium Assay Kit Fura-2 wash method Figure 1. Response of CHO-K1 endogenous P2Y receptor to ATP. CHO-K1 cells were plated overnight in 100 μl culture medium on two 96 well plates. The next day, one plate of cells was dye-loaded with 100 μl/well of 1X Dye-loading solution (BD TM Ratiometric Calcium Assay Kit), and the second one was loaded with 100 μl/well of Fura-2 AM. After 1 hour of incubation at 37 C, cells loaded with Fura-2 were washed three times with HBSS-Hepes buffer. The same amount of ATP was added to both plates by a FlexStation (Molecular Devices), and the data was recorded simultaneously. EC50 of ATP using BD TM Ratiometric Calcium Assay Kit is 129 nm. EC50 of ATP using Fura-2 wash method is 569 nm. A Histamine 300 nm B 0 Figure 2. Less well-to-well variation. HEK293 cells stably transfected with histamine H1 receptor were plated overnight in 100μl culture medium on two 96-well BD Biocoat poly-d lysine coated plates. The next day one plate of cells was dye-loaded with 100μl/well of BD Ratiometric Calcium Assay kit as recommended in the kit protocol (Panel A, Columns 1-3) and the other one was loaded with Fura-2 AM after removing the medium (Panel A, Columns 4-6). After 1 hour incubation at 37 C, cells loaded with Fura-2 were washed three times with HBSS- Hepes buffer. The same dilutions of histamine were added to both plates using a FlexStation (MDS), and the data was recorded simultaneously. Dose response curve was generated by SoftMaxPro (MDS). Panel A. Kinetic curves of calcium response to different concentrations of histamine. Cells were lost in several wells during washing procedures required for the Fura-2 wash method, as indicated by the lack or inconsistent Calcium response in the top 3 rows of the wells of columns 4-6. Panel B. Histamine dose response curve using BD Ratiometric Calcium Assay Kit. Dose response curve using Fura-2 wash could not be plotted because of poor data quality due to the cell loss. 644243 4/24/09 Page 3 of6
A B ATP 10μM 0 Figure 3. Single Cell Imaging Data on BD Pathway 855. CHOK1 cells were plated overnight in 100μl culture medium on a 96 well BD Falcon Imaging Plate (Cat No. 353219). The next day, the cells were dye-loaded with 100μl of BD Ratiometric Calcium Assay kit and 2.5mM probenecid at 37 C for one hour. Panel A. Kinetic curves of calcium response to different concentrations of ATP were measured by a BD Pathway 855. Panel B. Segmentation of the fluorescent signal into specific regions of interest (ROIs) by BD AttoVision software. 644243 4/24/09 Page 4 of6
CALCIUM FLUX ASSAY PROTOCOL Note. It is important to read the entire protocol before you start the experiment. CELL PREPARATION Cell number needs to be optimized for each assay. Optimal assay conditions require a confluent monolayer of cells prior to the assay. For adherent cells, it is usually recommended to plate 50K to 70K cells/well for a 96 well plate and 12K to 20K cells/well for a 384 well plate the day before the experiment. Add 100 μl/well of cell suspension to 96-well plates or 25 μl/well to 384-well plates, allow cells to attach and grow overnight for 16 to 24 hours in cell culture incubators. Prior to loading the cells with dye,, examine the cells in the plate wells for confluency and overall health. For non-adherent cells, dispense 100 μl/well of cells in culture medium to 96-well or 25 μl/well to 384-well poly-d Lysine or other ECM coated plates a couple of hours before the experiment. Allow cells to settle and attach to the bottom of the plates at room temperature. Prior loading the dye, centrifuge the plates briefly with brake turned off. PREPARATION of 1X DYE-LOADING SOLUTION 1. Remove a vial of Ratiometric Calcium Indicator from -20 C, and allow equilibrate at room temperature for 5 minutes.. Add 100 μl 100% DMSO, reconstitute the pellet by pipetting up and down several times. Note. It is critical to completely solubilize the Ratiometric Calcium Indicator: keep the reconstituted Calcium Indicator at room temperature for 10 minutes before the preparation of 1X Dye-loading solution. 2. To prepare 1X Signal Enhancer, transfer 10 ml of 10X Signal Enhancer to 90 ml of 1X Calcium Assay buffer, mix well. Note. For cells that require probenecid for loading (e.g. CHO), prepare fresh probenecid stock in 1N NaOH and then dilute the stock in 1X Signal Enhancer to a concentration of 5 mm.( CHO-K1 cells have high pump activity so adding probenecid would ensure that the dye is retained in the cell) 3. To prepare 1X Dye-loading Solution for one cell plate, pipette 10 ml of 1X Signal Enhancer to a separate tube, add 10 μl of Calcium Indicator to 10 ml of 1X Signal Enhancer, mix by inverting the tube several times. 4. Aliquot the unused Ratiometric Calcium Indicator to several microfuge tubes, seal tightly and store at -20 C.. 1X Signal Enhancer could be stored at room temperature. Note. The reconstituted Ratiometric Calcium Indicator is stable for at least one month if the tube is sealed tightly. For best result, place the tubes in a zip lock plastic bag with desiccant and avoid repetitive freeze-thaw cycles. CELLAND DYE SOLUTION PREPARATION FOR IMAGING APPLICATIONS: 1. For imaging applications, reduce the number of cells per well to 10K to 20K cells/well for a 96-well plate and 2.5 to 5K for a 384- well plate. Confluent cells samples may interfere with the accuracy of single cell measurements and imaging data analysis. BD Falcon Imaging Plate Flat Bottom plates are recommended for imaging experiments. 2. For the use of BD Ratiometric Calcium Assay Kit on imaging instruments, it is recommended to filter the 1X Dye-loading Solution before the dye loading step. DYE LOADING 1. Remove cell plates from incubator and add an equal volume of 1 X Dye-loading Solution to each well (e.g. 100 μl to 100 μl culture medium/well for 96-well plates, or 25 μl to 25 μl culture medium/well for 384-well plates). Note. The components of the kit do not have interference with serum or phenol red. However, for some assays that require a serum free environment, culture medium that contains serum should be removed prior to dye loading and replaced with an equal volume of 0.5X Dyeloading Solution. The 1X Dye-loading Solution is stable for 8 to 12 hours at room temperature and 2-3 weeks at -20 C. 2. Incubate cell plates with dye for 1 hour in cell culture incubator. Note. If the calcium flux assay is going to be performed at room temperature, place the cell plates at room temperature for at least 20 minutes to cool down before placing the plates on readers. If the calcium flux assay requires 37 o C, perform the assay immediately after dye-loading. 644243 4/24/09 Page 5 of6
CALCIUM FLUX ASSAY Place the cell plates on a FlexStation, FDSS or BD Pathway Bioimager 855 and perform calcium flux assay as described in instrumentation manuals. For assays performed on a FlexStation, use the following wavelength parameters: Excitation Emission Cutoff Lm1 340 510 455 Lm2 380 510 455 For assays performed on a BD Pathway 855 Bioimager, use following filter sets: Excitation Emission Dichroic Numerator 334/10 435LP 400CLP Denominator 380/10ND 435LP 400CLP For assays performed on an FDSS, use the standard filters for Fura-2 calcium assays on the instrument. Note. Dispense speed and height for compound additions need to be optimized for each assay. TROUBLESHOOTING GUIDE 1. Low baseline fluorescent signal Inspect the cell density and morphology under a microscope. Low cell density or unhealthy cells could result in low baseline signal. Check the storage of Ratiometric Calcium Indicator. Calcium Indicator needs to be stored in a tightly sealed tube frozen with desiccant. Once it is reconstituted, repetitive freeze thaw cycles should be avoided. Make sure that Ratiometric Calcium Indicator is mixed well with 1X Signal Enhancer before dye loading. 2. Response to agonist lower than expected Check the overall health of cells. Cell density is too high or too low. Cell number titration may be necessary. 3. Well-to-well variations after agonist addition. Optimize the dispense height and speed for ligand additions to ensure instant mixing. Cells should be evenly distributed among wells. Before plating, microscopically examine the culture to be sure that they have been broken up into single cells. Clumpy cells respond with greater variability. Check liquid handling system for accuracy of dispense. 4. Response from cells after buffer addition. Unhealthy cells could respond to buffer addition. Make sure cells are not over confluent or unhealthy before plating. Cells are disturbed by high compound addition speed. Set the pipetting speed and height properly to avoid stimulating the cells by physical forces during compound addition. 644243 4/24/09 Page 6 of6